In human cells, nucleocytoplasmic transport of macromolecules is mediated by 19 members of the Karyopherin2 (Kap2s/Importins/Exportins) protein family. Kap2s bind their transport substrates through distinct nuclear localization or export signals (NLSs or NESs), and transport them through the nuclear pore complex (NPC). The only known classes of NLS and NES are the short basic classical-NLS, the PY-NLS and the leucine- rich NES (LR-NES), recognized by Kap?/Kap?1, Kap?2 (or Transportin) and CRM1, respectively. Most other Kap?s recognize diverse sequences that occlude the identity of their signals. Nuclear transport is further complicated as most Kap?s can recognize multiple signals through multiple substrate binding sites. This proposal describes structural and biochemical analyses of substrate recognition by export-Kap? CRM1 and import factor Kap?2. CRM1 is the most general and versatile export-Kap?. Structural studies of CRM1-substrate complexes will reveal general principles for recognition of multiple NES classes and mechanistically explain different steps of nuclear export such as export complex assembly in the nucleus and substrate dissociation in the cytoplasm. Kap?2 imports numerous mRNA binding proteins into the nucleus. We have recently defined a new NLS in these proteins termed PY-NLS and uncovered 81 new potential Kap?2 substrates in a pilot bioinformatics initiative. Here, we propose structural and thermodynamics studies of Kap?2 interactions with diverse PY-NLSs to deepen biophysical understanding of PY-NLS recognition and to generate structural and thermodynamics training datasets for future computational work to accurately and comprehensively predict Kap?2 substrates in genomes. Public Health Relevance: This grant aims to discover how protein cargoes are recognized by nuclear export factor CRM1 to be exported from the cell nucleus. CRM1 is the most general and versatile nuclear export factor known to date and our work will inform on this basic cellular process as well as explain a process that when disrupted can result in heart disease and cancer. We will also study the reverse process of how protein cargoes are recognized by nuclear import factor Kap-beta2/Transportin.